Materials
siRNAs: Silencer Select GAPDH Positive Control siRNA was purchased from Thermo Fisher Scientific (San Jose, CA, USA). These siRNAs were used after labeling with Cy5 using the Label IT siRNA Tracker Cy5 Kit (Mirus Bio LLC, Madison, WI, USA). Peptides: The synthetic peptide Q-tag-R9-HA (5′-Acetyl-LLQGRRRRRRRRRYPYDVPDYA-COOH-3′) was purchased from Eurofins Genomics (Ebersberg, Germany). Cell lines: A431 human epidermoid carcinoma cells were obtained from the Cell Resource Center for Biomedical Research, Institute of Development, Aging, and Cancer, Tohoku University (Sendai, Japan). HEK293 cells were obtained from the Japanese Collection of Research Bioresources Cell bank (Osaka, Japan). These cells were cultured in RPMI 1640 medium with L-glutamine and sodium bicarbonate (liquid, sterile-filtered, suitable for cell culture; Sigma Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Biowest, France) and 1 × Anti-Anti (Gibco, Thermo Fisher Scientific, Waltham, MA, USA).
Construction of expression vector
The gene encoding anti-EGFR nanobody from Ia1 llama antibody was amplified to fuse the K-tag-His-tag (GGGGSMRHKGSHHHHHH), R9-His-tag, (GGGGSRRRRRRRRRHHHHHH), or Cmyc′-His-tag (TMFLISEEDLQHHHHHH) to its C-terminus by polymerase chain reaction (PCR). PCR products and pRA expression vector were digested with NcoI and SpeI in case of K-tag-His-tag and R9-His-tag, or with NcoI and SacII in case of Cmyc′-tag, and ligated to each other at 16 °C for 30 min to generate pRA-Nb-K-His, pRA-Nb-R9-His, and pRA-Nb-Cmyc′-His expression vectors.
The gene encoding MTG from Streptomyces mobaraensis (accession number DQ132977) fused 6 × His-tag to the C-terminus synthesized by Thermo Fisher Scientific and was amplified by PCR to generate NcoI and EcoRI sites. PCR products and the pET22b expression vector were digested with NcoI and EcoRI, followed by ligation at 16 °C for 30 min to generate the pET22b (+)-MTG-His expression vector.
Expression and purification of recombinant antibodies
Escherichia coli BL21 (DE3) transformants harboring pRA-Nb-K-His, pRA-Nb-R9-His, and pRA-Nb-Cmyc′-His were cultured at 28 °C in flasks containing 2 × YT medium supplemented with 100 µg mL−1 ampicillin, and protein expression was induced by adding 1 mM IPTG when the absorbance of the culture at 600 nm reached 0.8. After incubation at 28 °C for 16 h, the culture supernatant and intracellular soluble and insoluble fractions were collected and evaluated by SDS-PAGE and western blot. Proteins were purified using IMAC (Ni Sepharose 6 Fast Flow; GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and SEC (HiLoad 26/600 Superdex 75 prep grade; GE Healthcare Bio-Sciences AB).
Expression and purification of MTG
Transformed E. coli BL21 (DE3) cells harboring expression plasmids encoding MTG were incubated in 2 × YT medium containing 100 g mL−1 ampicillin at 37 °C, and expression of recombinant antibodies under the control of the T7 promoter was induced by adding 0.4 mM IPTG when the absorbance of the culture at 600 nm was 0.8. After additional incubation at 20 °C for 30 h, the bacterial supernatant was collected and purified using IMAC and SEC (HiLoad 26/600 Superdex 200 prep grade; GE Healthcare Bio-Sciences AB).
MTG-mediated fusion of Nanobody-K and Q-R9 and purification of Nanobody-R9MTG
The fusion of each nanobody and R9 peptide was conducted by mixing 5 µM Nanobody-K (or Nanobody-Cmyc′), 5–35 µM Q-R9, and 0.03 U mL−1 MTG in PBS (pH 7.4) at 20 °C for 6 h. The reaction product was analyzed by SDS-PAGE and western blot analysis, and reaction efficiency was evaluated based on band intensity using Image Quant Las 4000 (GE Healthcare Bio-Sciences AB). The reaction product was purified using IMAC (1 mL His trap HP; GE Healthcare Bio-Sciences AB) with 0–400 mM imidazole gradient in 1 × PBS containing 750 mM NaCl to remove MTG and unreacted Q-R9 and Nanobody-K.
Electrophoretic mobility shift assay
Nanobody-R9MTG and 5 pmol Cy5-siRNA were mixed, with increasing amount of Nanobody-R9 in PBS. The mixture was incubated at 4 °C for 30 min and then electrophoresed on 2% (w/v) agarose gels in TAE buffer. The mobility shift of the siRNA band was visualized on Cy5 detected using a UV-transilluminator.
Flow cytometry
The specific binding of Nanobody-R9MTG or Nanobody-K to EGFR on the cell surface was analyzed by flow cytometry. For this, forty-seven nM of Nanobody-R9MTG or Nanobody-K were labeled with Fluorescein Labeling kit-NH2 (Dojindo, Inc., Kumamoto, Japan) and mixed with 2 × 106 EGFR-positive A431 cells. The mixtures were incubated for 60 min on ice. The cells were washed three times with PBS containing 0.01% bovine serum albumin and analyzed by flow cytometry (FACS Accuri 6; BD Biosciences, Franklin Lakes, NJ, USA).
Confocal microscopy
A431 cells (1.5 × 105) were grown on Glass bottom dishes (Matsunami Glass Ind., Ltd., Japan) in RPMI medium for 24 h at 37 °C and 5% CO2. Then, 50 pmol Cy5-siRNA was mixed with 300 or 1500 pmol Nanobody-R9MTG in PBS (final volume 500 µL), and the mixture was incubated at 4 °C for 60 min. Lipofectamine 2000 (Thermo Fisher Scientific, Japan) was used as a positive control. The reaction mixture was added to the culture medium of A431 cells followed by incubation at 37 °C for 6 h in the cell incubator. The cells were washed twice with PBS and stained with 1 mg mL−1 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, Japan) and Cell Mask Green plasma Membrane stain (FITC detection, Thermo Fisher Scientific, Japan). After staining, the cells were observed under a confocal microscope (FV1200-D; Olympus, Japan).
Western blot
A431 cells (1.5 × 104) were grown on 24-well plates in RPMI medium for 24 h at 37 °C. Then, 20 pmol Cy5-siRNA was mixed with 120 or 6000 pmol Nanobody-R9MTG in PBS (final volume 150 µL) and incubated at 4 °C for 60 min. Lipofectamine 2000 was used as a positive control. The mixture was added to the culture medium of A431 cells and incubated at 37 °C for 72 h in a cell incubator. Thereafter, the cells were washed in PBS and lysed directly with RIPA buffer (Nacalai Tesque, Kyoto, Japan). The extracted proteins were then transferred onto nitrocellulose membranes (Millipore, Bedford, MA, USA) that were incubated with primary antibodies against GAPDH (1:7500; MBL, Nagoya, Japan) and β-actin (1:7500; MBL), followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse Ig secondary antibody (1:7500; Proteintech Group, Inc., Tokyo, Japan). Finally, Chemi-Lumi One (Nacalai Tesque) was used to visualize the protein bands.
Real time PCR
First, 120 or 6000 pmol Nanobody-R9MTG was mixed with 20 pmol siRNA in 150 µL of PBS and incubated at 4 °C for 60 min. A431 cells (3.0 × 104) were grown on 24-well plates (Costar 24 well 3524, Corning, NY, USA) in RPMI medium at 37 °C overnight. The cells were then washed in PBS and incubated with 250 mL Nanobody-R9 siRNA (with a final concentration of 40 nM siRNA) in 250 µL RPMI medium at 37 °C for 24 h. Thereafter, cells were washed three times in PBS. Next, the cells were lysed using SingleShotCell Lysis kit (Bio-Rad laboratories, Inc., CA, USA) and cDNA was synthesized using Advanced cDNA synthesis kit for RT-PCR (Bio-Rad laboratories, Inc.) according to the manufacturer’s instructions. RT-PCR of GAPDH transcripts was performed using a mixture of 1 µL cDNA, 5 µL 5 × iScript SYBR Green supermix (Bio-Rad laboratories, Inc.), 10 pmol µL−1 GAPDH forward primer: 5′-GTCTCCTCTGACTTCAACAGCG-3′ and GAPDH reverse primer: 5′-ACCACCCTGTTGCTGTAGCCAA-3′ and nuclease-free water by thermal cycling under following conditions: 40 cycles of 95 °C for 30 s, 95 °C for 10 s, and 60 °C for 30 s. The cDNA content was calculated using the ΔΔCt method with Actin as a housekeeping gene (Actin forward primer: 5′-CACCATTGGCAATGAGCGGTTC-3′ and Actin reverse primer: 5′-AGGTCTTTGCGGATGTCCACGT-3′.
